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Variety diagnostic PCR-RAPD markers for aromatic rice varieties grown in Eastern Part of India
Anuprita Ray, Arpita Pattanaik, K.C.Samal, S. S. Kshirsagar and G. R. Rout* Dept. of Agricultural Biotechnology, Orissa University of Agriculture and Technology, Bhubaneswar-751003, Odisha, India. Tele fax-0091-674-2397755 *Email- grrout@rediffmail.com

Running Title: Ray et al., ------- Genetic variability of aromatic rice through RAPD markers Abstract PCR-RAPD diagnostic marker was used to determine the genotypic identification and genetic variation in 50 aromatic rice varieties grown in Eastern part of India. Out of thirty primers, 12 primers showed DNA amplification and polymorphism among the 50 rice varieties. A total of 104 bands appeared by using 12 decamers in 50 aromatic rice varieties. Out of which, 95 bands are polymorphic and three are unique bands. These bands are varietal specific diagnostic markers used for identification and also for protection of plant varieties by registration. The results revealed that all the tested primers showed distinct polymorphism among the varieties indicating the robust nature of RAPD markers. Most of the primers showing the highest polymorphic information content (PIC) and resolving power (Rp). The cluster analysis indicate that the aromatic rice genotypes are grouped into two major clusters. Among the two major clusters, one major cluster had only two varieties and second major cluster having 47 varieties. Based on this study, the larger range of similarity values using RAPD markers provides greater confidence for the assessment of genetic relationships among the varieties. The information obtained from the RAPD profile helps to identify the variety diagnostic markers in 50 aromatic rice genotypes. Significant genetic variation at maximum number of loci between varieties indicates rich genetic resources in rice. The intra and inter genetically variation might be useful for breeders to improve the aromatic rice varieties through selective breeding and cross breeding programs. Key words: Aromatic Rice, DNA profile, Diagnostic PCR-RAPD marker, Polymorphism.

Introduction Rice (Oryza sativa) belonging to the family Poaceae and subfamily Oryzoidea is the staple food for one third of the worlds population and occupies almost one-fifth of the total land area covered under cereals. It is grown under diverse conditions and over wide geographical range. Most of the worlds rice is cultivated and consumed in Asia, which constitutes more than half of the global population. Approximately 11% of the worlds arable land is planted annually to rice, and it ranks next to wheat. The worlds rice production has doubled during last 25 years, largely due to the use of improved technology such as high yielding varieties and better crop management practices (Byerlee, 1996). Further scope of crop improvement depends on the availability genetic diversity and variability and use of new biotechnological tools. There is a rich diversity exist in rice. India is a leading country in the export of promising rice varieties including scented rice. Aromatic rice is an important commercial commodity. It is more preferred by the consumers all over the world because of its scent and palatability. There is a strong need that the germplasm of this cash crop be collected preserved and characterized in details. India has enacted a legislation as Protection of plant varieties and Farmers Right Act, 2001 for protection of plant varieties by registration. Both DUS (distinctiveness, uniformity,stability) and molecular characteristics are essential to help the identification of basumati rice varieties (Patra and Chawla, 2010). Indian rice varieties have been developed traditionally by selections, hybridization and back crossing with locally adapted high yielding lines. The number of parental lines used in the breeding programs is however quite small, resulting in a narrow genetic base. Selection of varieties based on morphological characters are not very reliable because major characters have low heritability and are genetically complex. Many Indian farmers are still growing local stains under different names and they also bring some varieties from distant places. There is a strong need to collect this germplasm and identify the genotypes. Molecular markers based DNA sequence is found to be more reliable (Virk et al., 1996). They represent an opportunity to provide information on the variation that exists in a particular species within a local region. Molecular markers provide information that helps in identifying the genotypes and their association with close relatives and phylogenetic relationships. There are many reports that RAPD markers are unbiased and neutral markers for genetic diversity study, genetic mapping, population genetics as well as genetic diagnostics in

3 plants. The PCR-based RAPD approach using single decamer arbitrary primers requires less amount of DNA and is technically simple as compared to other molecular markers. It can provide the assessment of genetic distances, seed purity and resolution of uncertain parentage. RAPD markers have been used in the analysis of rice genotypes by various researchers (Raghunathachari et al., 2000; Rahman et al., 2007; Bhuyan et al., 2007 Ray Choudhury et al., 2001, Patra & Chawla, 2010). However, collection of Indian aromatic rice varieties remain unexplored as there are very scanty references available (Ray Choudhury et al., 2001). Keeping in view, the present investigation was undertaken to estimate variety diagnostic markers for identification and phylogenetic relationship among 50 varieties of aromatic rice grown in Eastern part of India by using RAPD markers. Materials & Methods Plant Materials: Fifty elite aromatic rice varieties were collected from the germplasm centre maintained by the department of Plant Breeding and Genetics, Orissa University of Agriculture & Technology, Bhubaneswar for PCR-RAPD analysis. All the varieties have been categories into the germplasm accession number (Sl. No. 1 to 50 as per table 1). The morphological and agronomic characteristics have also been indicated in Table 1. The seeds were shown in the earthen pots and kept in the green house for germination. Leaf samples were collected and subsequently stored at 20C for isolation of genomic DNA. Genomic DNA Extraction Genomic DNA was extracted from young leaves using N-Cetyl-N,N,Ntrimethylammonium bromide (CTAB) method described by Doyle and Doyle (1990) with modifications. Two grams of fresh leaf material were washed in distilled water and subsequently rinsed with 80% (v/v) ethanol and then grounded in liquid nitrogen. Ten milliliters of preheated extraction buffer [4 % (w/v) CTAB, 0.2% -mercaptoethanol (v/v), 100 mM Tris-HCl pH 8.0, 20 mM EDTA pH 8.0, 1.4 M NaCl ] were then added per 2 g of leaf powder material and incubated for two hours at 65 0C. The lysate was purified with chloroform : isoamyl alcohol (24:1). The DNA pellet was resuspended in 200 l to 300 l of Tris-EDTA buffer (10 mM Tris HCl, 1 mM EDTA, pH = 8.0). DNA was reprecipitated by adding 80% ethanol in the presence of 0.3M sodium acetate, and palleted by centrifugation. The pelletes were lyophilized

4 and resuspended in TE buffer. The RNA was removed by RNAse treatment at 37 0C for 1 hour. For further purification, DNA solution was extracted once with equal volume of phenol and chloroform : isoamyl alcohol (24:1) followed by two extractions with chloroform : isoamyl alcohol (24:1). The upper aqueous phase was separated after centrifugation and mixed with 1/10th volume of 3M sodium acetate. DNA was precipitated by adding two adding two volumes of chilled absolute alcohol, pelleted, dried in vacuum and dissolved in TE buffer. Quantification of DNA was accomplished by analyzing the purified DNA on 0.8% (w/v) agarose gel electrophoresis alongside diluted uncut lambda DNA as standard. DNA was further diluted with TE to a concentration of 20ng/l for use in PCR analysis. Primer selection Initially, thirty decamer primers (M/S Bangalore Genei, India) were evaluated for ten randomly chosen genotypes to test their suitability in amplifying aromatic rice. Primers were selected on the basis of intensity of bands, repeatability of markers of genotypes. Finally, twelve primers were selected for the analysis of 50 aromatic rice genotypes (Table 2). PCR amplification & electrophoresis Polymerase chain reactions (PCR) was carried out in a final volume of 25 l containing 20 ng template DNA, 100 M of each deoxyribonucleotide triphosphate, 20 ng of primer 1.5 mM MgCl2, 1x Taq buffer (10 mM Tris-HCl [pH-9.0], 50 mM KCl, 0.01% gelatin), and 0.5 U Taq DNA polymerase (M/S Bangalore Genei, Bangalore, India). Amplification was performed in a thermal cycler (Pelican, India) programmed for a preliminary 2 min denaturation step at 94 0C, followed by 40 cycles of denaturation at 94 0C for 20 sec., annealing at 37 0C for 30 sec. and extension at 72 0C for 1 min, finally at 72 0C for 10 min amplification. The details of primers used were presented in Table 2. Amplification products were separated alongside a molecular weight marker (1.0 Kb plus ladder, M/S Bangalore Genei, Bangalore, India) by 1.2 % agarose gel electrophoresis in 1x TAE (Tris Acetate EDTA) buffer stained with ethidium bromide and visualized under UV light. Gel photographs were scanned through Gel Doc System (Gel Doc. 2000, BioRad, California, USA) and the amplification product sizes were evaluated using the software Quantity one (BioRad, California, USA).

Data analysis

5 RAPD reactions were performed twice for each primer with DNA sample of each genotype and only reproducible bands were included in the study. Furthermore, gel images of the genotypes were carefully and independently scored by two persons. Amplifications were scored as discrete variables, using 1 to indicate presence and 0 for absence of band. A binary matrix was obtained by visual scoring of the bands. Efficiency of discrimination was assessed in terms of the number of polymorphic markers generated and the ability to generate unique band. Pairwise-similarity matrices based on RAPD data were determined using Jaccards similarity coefficient (Sneath and Sokal, 1973). The average of similarity matrices was used to generate a tree by UPGMA (Unweighted Pair-Group Method Arithmetic Average) using NTSYSPC, version 2.0 (Rohlf 1995). Results and Discussion The present investigation offers an optimization of primer screening for evaluation of genetic relationship of 50 varieties of aromatic rice through RAPD markers. Ten genotypes were used for screening primers obtained B- and C-series primers produced relatively OPB-12, OPC-08 and OPA-08 from different series for amplification by using polymerase chain reactions. The results showed that A-, more amplification fragments compared to N- and D-series decamer primers. The amplification generated by primers produced maximum number of DNA fragments; the size of the DNA fragments ranged from 200 to 2500 base pairs. Primer OPA- 08 amplified 14 fragments whereas; OPB-12 produced 11 bands (Table 2). It was also noted that some of the primers did not show any amplification by using the ten rice genotypes. The twelve decamers produced good amplification of RAPD fragments. Among the thirty primers, twelve primers were selected to analyze the genetic relationship among the 50 aromatic rice genotypes through RAPD markers. The reproducibility of the amplification product was tested with two independent extractions. Most of the amplification reactions were duplicated. Only bands that were consistently reproduced across amplifications were considered for the analysis. Bands with the same mobility were considered as identical fragments, receiving equal values, regardless of their staining intensity. When multiple bands in a region were

6 difficult to resolve, data for that region of the gel was not included in the analysis. As a result, twelve informative primers were selected and used to evaluate the degree of polymorphism within 50 varieties of aromatic indica rice under family Poaceae and subfamily Oryzoidea. The maximum and minimum number of bands were produced by the primers OPA-08 and OPC-07 respectively (Table 2). A total of 109 amplified fragments was scored across the 50 varieties of aromatic rice for the selected primers, and was used to estimate genetic relationships among themselves. The patterns of RAPD produced by the primers OPC-08, OPB-12 and OPA-08 are shown in the Figures 1A-C. The genetic variation through molecular markers has been highlighted in a number of rice genotypes (Raghunathachari et al., 2000; Ray Choudhury et al., 2001; Rahman et al., 2007). The genetic similarity as determined by RAPD fingerprinting also corresponded considerably with the known pedigrees. The similarity matrix was obtained after multivariate analysis using Nei and Lis coefficient (data not shown). The similarity matrix was then used to construct a dendrogram with the UPGMA method (Figure 2). The dendrogram shows two major clusters within 50 varieties of aromatic indica rice. Among the two major clusters, one major cluster had only two varieties (Kukudajata and Manasi) and other major cluster divided into two minor clusters. First minor cluster had only one variety i.e. ``Kalajeera`` and second minor cluster having 47 variety. Second minor cluster again divided into two sub-minor clusters; first sub-minor cluster having twenty two varieties. Among the twenty two varieties, ``Jalaka``, ``Dangarbasumati`` and ``Gangabali`` having 100 % similarity among themselves in particular with panicle length. The variety ``Basumati`` and ``Basumati-1`` making same cluster with 65% similarity in days to flowering and panicle length. The variety ``Gopalabhog`` and ``Kalikati`` having 84% similarity with respect to days to flowering, panicle length and panicle number. The variety ``Basnasapuri`` ``Sagadadhuli`` having 80% similarity among themselves and also 52% similarity with variety ``Kalajera``. The second sub minor cluster having twenty five varieties and divided into two clusters. First cluster

7 having four varieties with 30 to 50% similarity among themselves (``Nanu``, ``Sirimula``, ``Thakurabhoga`` and ``Jalaka``). Second cluster having 21 varieties with two sub clusters. First sub cluster having two varieties i.e. ``Basumatidhan`` and ``Dulhabhog``. Second sub cluster having 19 varieties and divided into two clusters. One cluster having one variety ``Kaminibhog-2`` with 30% similarity with other 18 varieties. Second cluster again subdivided into two clusters i.e. one having 8 varieties and others making two clusters. One having 6 varieties Local-2``, (``Sujata``,``Heerakani``,Sheetabhog``,``Ganjam

``Tulasiphulla`` and ``Kalikati``) and other having 4 varieties (``Nalidhan``, ``Nuadhusura``, ``Thakurasuna`` and Dhoiabankoi) with more than 50% similarity among themselves. Molecular markers show better resemblance with the pedigree as compared to morphological markers. Patra and Chawla (2010) reported that the biochemical and RAPD molecular markers helps to establish the distinctiveness of basumati rice varieties. Genetic variation is important in maintaining the developmental stability and biological potential of the genotype. The results indicate that there was very close variation among the varieties. These results suggest that the use of different RAPD primers would enable to asses the genetic diversity of aromatic rice variety as reported earlier in other variety of rice (Blair et al., 1999). Joshi et al (2000) studied the genetic diversity and phylogenetic relatedness in Oryza by ISSR markers. The present study showed that the higher percentage of polymorphism as compared with other molecular marker as reported earlier (Galvan et al., 2003, Mohapatra et al., 2005). Bhuyan et al (2007) illustrated the genetic diversity in traditional lowland rice grown in Assam using both RAPD and ISSR markers. Further, Youssef et al (2010) used both RAPD and ISSR markers to identify the new promising drought tolerant lines of rice under drought stresses. These traits with molecular differences commented upon in this investigation suggest that these rice varieties were belongs to indica rice with introgressions from wild rice land races. Significant genetic variation at maximum number of loci between varieties indicates rich genetic resources in rice. The intra and inter genetically variation might be useful for breeders to improve the programs. aromatic rice through selective breeding and cross breeding

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Acknowledgement

The authors wish to acknowledge to Department of Biotechnology, Government of India for providing funding for student research under PG HRD program. References Bhuyan N., Borah BK., Sarma RN. (2007) Genetic diversity analysis in traditional lowland rice (Oryza sativa L.) of Assam using RAPD and ISSR markers. Current Sci., 93:967-972. Blair MW, Panaud O, McCouch SR (1999). Inter-simple sequence repeat (ISSR) amplification for analysis of microsatellite motif frequency and fingerprinting in rice (Oryza sativa L.). Theor. Appl .Genet. 98: 780-792. Byerlee D (1996). Knowledge-Intensive Crop Management Technologies: Concepts, Impacts, and Prospects in Asian Agriculture. International Rice Research Conference, Bangkok, Thailand, 3-5 June, 1996. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 1315 Joshi SP, Gupta VS, Aggarwal RK, Ranjekar PK, Brar DS (2000) Genetic diversity and phylogenetic relationship as revealed by Inter simple sequence repeat polymorphism in the genus Oryza. Theor. Appl. Genet. 100: 1311-1320. Mohapatra A, Rout GR (2005) Identification and analysis of genetic variation among rose cultivars using random amplified polymorphic DNA. Z Naturforschung 60C: 611617 Patra N. , Chawla HS (2010) Biochemical and RAPD molecular markers for establishing distinctiveness of basumati rice (Oryza sativa L.) varieties as additional descriptors for plant variety protection. Indian Journal of Biotechnology, 9:371-377. Rahman SN., Islam MS., Alam MS., Nasirudin KM (2007) Genetic polymorphism in rice (Oryza sativa L.) through RAPD analysis. Indian Jour Biotechnology, 6: 224-229. Raghunathachari P., Khanna VK., Singh US, Singh NK (2000) RAPD analysis of genetic variability in Indian scented rice germplasm (Oryza sativa L.). Current Sci., 79 (7): 994998. Ray Choudhury, P., Kohli S., Srinivasan K., Mohapatra T., Sharma RP (2001) Identification and classification of aromatic rices based on DNA fingerprinting. Euphytica, 118:243-251. Rohlf FJ (1995) NTSYS-PC Numerical taxonomy and multivariate analysis system. Version 1.80, Exeter Software, Setauket, New York Sneath, P.H.A., and R.R. Sokal. (1973) Numerical taxonomy: The principles and practice of

9 numerical classification. W.H. Freeman, San Francisco, CA. Virk PS., Ford-Lloyd BV., Jackson MT., Pooni HS., Clemeno TP., Newbury HJ (1996) Predicting quantitative variation within rice germplasm using molecular markers. Heredity, 76: 296-304. Youssef MA., Mansour A., Solliman SS (2010) Molecular markers for new promising drought tolerant lines of rice under drought stress via. RAPD-PCR and ISSR markers. Jour. of American Sci., 6: 355-363.

Table 1. Morphological characteristics of aromatic rice varieties used for molecular analysis.
Accessio n no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Name of Genotypes Days To flowering Plant Height (cm) Panicle Length (cm) Panicle Number No of Fertile grains 1000 grain weight (g) Harves t index

Fertility (%)

Potential Yield(q/ha)

Nanu Basmatidhan Basmati Basumati-1 Dholabankoi Jalaka Kalikati Dulhabhoga Kanakachampa Gopalabhoga Jala Dangarabasamati Kaminibhoga-2 Kalajeera

103 91 100 100 102 104 101 100 98 102 103 101 103 104

133.9 122.4 120.0 103.3 127.1 107.4 133.1 125.2 135.6 121.5 115.4 126.0 129.0 129.9

26.6 22.3 26.7 24.1 27.1 24.7 24.9 23.0 24.5 24.6 24.5 22.8 25.2 25.6

9 6 8 9 9 6 7 7 7 7 10 7 7 8

165 116 109 62 169 112 164 141 146 125 136 135 145 123

76.6 88.2 74.1 65.9 86.5 89.6 92.9 85.3 76.2 78.0 75.7 82.3 82.3 89.9

11.0 12.8 11.8 16.9 12.4 14.2 10.9 18.9 12.1 11.5 11.6 15.6 11.9 14.4

0.36 0.37 0.43 0.21 0.37 0.31 0.34 0.41 0.38 0.39 0.33 0.39 0.43 0.38

17.48 18.62 23.95 23.95 29.42 22.81 21.39 17.65 18.62 18.57 18.71 19.20 19.77 25.55

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Accessio n no. 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 Name of Genotypes Days To flowering Plant Height (cm) Panicle Length (cm) Panicle Number No of Fertile grains 1000 grain weight (g) Harves t index

Fertility (%)

Potential Yield(q/ha)

Kalikati-1 Badsahbhog Ganjam local-1 Karpurakranti Basnaparijat Basnasapuri Saragadhulli Magura Kalajeera Lajakulibadan Kalajauvan Kukudajata Gatia Jaiphulla Gangaballi Manasi Chatianaki-1 Thakursuna Bishnubhog Dhobaluchi Srimula Ganjam local-2 Thakurbhog Nuakalajeera Pimpudibasa Heerakani Chatianaki Baranamgomati Nalidhan Sheetabhog Nuadhusura Tulasiphulla Sujata Neelabati Chinikamini Khosakani

102 104 98 102 104 104 103 108 100 103 101 103 107 102 97 101 99 103 100 91 105 105 106 105 100 98 98 104 105 103 104 98 96 108 100 104

148.2 135.9 123.0 130.9 117.6 145.2 129.5 111.7 125.6 136.0 140.3 136.2 136.2 105.0 125.4 115.4 140.7 108.1 129.8 103.9 125.9 130.0 127.4 136.1 124.5 117.6 147.2 147.9 141.1 120.6 125.3 110.8 115.3 110.6 126.2 127.8

27.5 27.4 23.9 24.5 24.3 25.5 25.6 21.90 25.0 26.7 29.8 26.2 23.2 20.6 25.0 24.6 29.0 26.8 22.8 18.8 25.2 24.5 28.8 27.2 26.4 25.2 29.5 25.4 27.7 23.6 25.5 23.2 24.7 26.9 28.0 27.4

6 9 8 7 10 11 10 7 7 9 4 9 8 6 9 9 7 6 7 7 8 7 10 7 6 10 6 8 13 8 8 9 9 7 8 7

153 159 137 88 147 173 132 112 125 150 143 140 135 107 139 117 123 185 195 123 131 128 145 159 160 175 126 97 174 145 110 108 140 160 119 174

69.0 90.6 86.5 74.6 91.5 79.4 82.0 83.0 85.6 80.4 90.9 79.6 82.3 83.7 93.9 94.4 72.0 84.2 95.9 83.6 76.4 87.0 89.9 97.8 85.9 88.0 71.9 88.9 93.1 84.4 75.7 79.4 90.0 86.2 81.0 87.3

11.2 11.5 11.8 13.6 11.1 11.0 11.8 17.6 13.9 12.5 13.5 12.4 18.0 19.2 11.6 13.2 12.5 11.5 13.3 23.4 11.1 13.9 12.9 11.0 13.3 11.0 12.8 19.8 12.3 10.7 15.9 13.7 14.1 14.0 12.3 13.9

0.36 0.33 0.39 0.39 0.34 0.38 0.42 0.30 0.40 0.37 0.31 0.43 0.39 0.41 0.41 0.40 0.38 0.37 0.39 0.57 0.31 0.40 0.33 0.32 0.37 0.40 0.41 0.41 0.37 0.40 0.41 0.49 0.51 0.34 0.39 0.37

21.54 24.48 19.20 17.65 22.22 25.07 19.23 17.06 18.47 28.42 18.60 19.59 24.37 25.68 24.48 23.48 19.59 28.99 27.36 27.46 21.59 27.29 28.12 20.78 18.57 23.16 18.84 22.61 24.07 19.13 20.72 10.49 26.32 23.45 18.04 28.41

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Table 2. DNA profile of 50 genotypes of aromatic rice by using RAPD primers.

12
Low Freque ncy band (530%) 6

Prime r Code

Sequence 5

Total no. of of bands

Polymo rphic band

Uniq ue band

Rare band (< 5%)

High Frequ ency band >30% 3

%age Polymorp hism

Size Range (bp)

Resolvi ng Power (Rp)

Avera ge PIC Value

OPC-8 OPB12 OPA-8 OPA-1 OPA-2 OPA-3 OPA-4 OPA-5 OPC07 OPC15 OPD08 OPD10

TGGACCGG TG 3 5 CCTTGACG CA 3 5 GTGACGPA GG 3 5 CAGGCCCT TC 3 5 TGCCGAGC TG 3 5 AGTCAGCC AC 3 5 AATCGGGC TG 3 5 AGGGGTCT TG 3 5 GTCCCGAC GA 3 5 GACGGATC AG 3 5 GTGTGCCC CA 3 5 GGTCTACA CC 3

11

11

100

3001700 2152500 2002500 230 1500 320 2500 375 2200 320 1900 260 1350 3202850 3903450 3403150 500 2700

4.8

0.91

11 14 10 9 7 9 7

11 14 9 7 5 6 7

1 0 0 0 0 1 0

1 1 0 1 0 0 0

8 10 7 6 3 5 4

1 3 2 0 2 0 3

100 100 90.00 77.78 71.43 66.67 100.00

2.68 5.64 3.12

0.98 0.88 0.512

2.98 0.318 3.90 3.12 0.448 3.87 0.437 0.412

66.67

3.76

0.384

71.43

3.98

0.436

87.50

2.95

0.378

10

90.00

4.40

0.570

13

14

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